The timely detection of diseases and the accurate identification of pathogens require the development of efficient and reliable diagnostic methods. In this study, we have developed a novel specific multivariate probe termed MRTFP (multivariate real-time fluorescent probe) by assembling strand exchange three-way-junction (3WJ) structures. The 3WJ structures were incorporated into a four-angle probe (FP) and a hexagonal probe (HP), to target the multivariate genes of Salmonella. The FP and HP enable single-step and multiplexed detection in RT-LAMP (real-time loop-mediated isothermal amplification) with exceptional sensitivity and specificity. Encouragingly, real food samples contaminated with Salmonella (Salmonella enteritidis and Salmonella typhimurium) can be readily identified and distinguished with a minimum detectable concentration (MDC) of 103 CFU/mL without the need for further culture. The introduction of MRTFP allows for simultaneous detection of dual or three targets in a single tube for LAMP, thereby improving detection efficiency. The MRTFP simplifies the design of robust multivariate probes, exhibits excellent stability, and avoids interference from multiple probe units, offering significant potential for the development of specific probes for efficient and accurate disease detection and pathogen identification.